The present invention relates to a genomic DNA fragment of the bacterium Streptococcus pneumoniae, a probe capable of specifically hybridizing with the genomic DNA of Streptococcus pneumoniae, a primer for specifically amplifying the genomic DNA of Streptococcus pneumoniae, a reagent and a method for selectively detecting, in a biological sample, said bacterium, used with the probe of the invention.
Research studies have been carried out on the isolation of two nucleotide fragments, on their sequencing, their specificity towards the genomic DNA of Streptococcus pneumoniae and their use for the production of hybridization probes intended for a diagnostic method.
These two fragments are respectively the hexB gene, deposited and accessible at the gene library of EMBL (European Molecular Biology Laboratory, Heidelberg, Germany) under the number M29686 and the study of which was especially the subject of the publication by M. PRUDHOMME, B. MARTIN, V. MEJEAN and J. P. CLAVERYS (1989) J. Bacteriol. 171, 5332-5338 and which encodes an essential protein of the system for the mismatch repair of the Streptococcus pneumoniae DNA, and the ami operon, deposited and accessible at the gene library of EMBL under the number X17337 and the study of which has especially been the subject of the publication by G. ALLOING, M. C. TROMBE and J. P. CLAVERYS (1990 Mol. Microbiol., 4, 633-644, and which is involved in the transport of oligopeptides in pneumococcus.
For each of these two fragments, various sub-fragments were prepared and used as hybridization probes for the genomic DNA of Streptococcus pneumoniae. The most commonly used probes are, in the case of the hexB gene, the hexB-S.sub.7 fragment obtained by the action of the restriction enzymes HindIII-BglIII (from nucleotide 1321 to nucleotide 1776) containing 455 nucleotides, and, in the case of the ami operon, the ami-S.sub.2 fragment obtained by the action of the restriction enzymes BamHI-EcoRI (from nucleotide 2419 to nucleotide 3564), containing 1145 nucleotides.
Each of these two probes was subjected to hybridization experiments according to the so-called "dot-blot" technique according to MANIATIS et al. (1982), Molecular Cloning, Cold Spring Harbor, with the genomic DNA of Streptococcus pneumoniae and the genomic DNA of other genera and species of bacteria, under stringent conditions (50% formamide, at 42.degree. C.), on nylon membranes (trade name Biodyne A, from the company Pall), using two concentrations of respectively 10 ng and 100 ng of genomic DNA.
Identical results were obtained with the two probes hexB-S.sub.7 and ami-S.sub.2. FIG. 1 shows the results obtained with the probe ami-S.sub.2 previously labeled by radioactive labeling with .sup.32 P (trade name Kit Multiprime from the company Amersham), the hybridizations being visualized by autoradiography, for 12 hours at -70.degree. C. According to FIG. 1, two DNA spots of respectively 10 ng (dotted arrow) and 100 ng (solid arrow) were prepared for each of the bacterial strains used. The bacterial strains were grouped together by series and numbered within each series as follows:
a series:
a1-a11: Clinical isolates of Streptococcus pneumoniae belonging to different serotypes.
b series:
b1-b5: Streptococcus oralis of the API collection (BioMerieux SA) (internal references API No. 7902025, 7902072, 8305023, 8040010, accessible at National Culture Type Collection under the reference NCTC11427, 8408077).
b6-b10: Clinical isolates classified Streptococcus sanguis based on API-20 Strep tests (BioMerieux SA) of which the result is indicated in brackets below in the order, SI (4061440), SII (0260451), SII (0270441), SI (0061440), SII (0240440).
b11-b12: Clinical isolates classified Streptococcus mitis based on the API-20 Strep tests (0040401 for both strains).
b13-b14: Clinical isolates classified Streptococcus milleri based on the API-20 Strep tests (1061010 for both strains).
b15-b16: Clinical isolates classified Streptococcus salivarius based on the API-20 Strep tests (5060451 and 5060461).
b17-b20: Clinical isolates of Enterococcus faecalis, Listeria monocytogenes, Haemophilus influenzae and Neisseria meningitidis.
c series:
c: "Atypical streptococci" obtained from clinical isolates.
According to FIG. 1, the results are as follows:
All the strains of Streptococcus pneumoniae of the a) series give very visible signals which are proportional to the concentration considered.
The atypical streptococci of the c, c91, c120, c108, c108, c92, c188, c139, c155, c184, c115, c65 and c174 series give the same signals as those of the a) series and could therefore be classified, based on this test, in the Streptococcus pneumoniae species.
All the strains of Streptococcus oralis of the b) series, with the exception of the b5 strain, the strain Streptococcus mitis b11 and the atypical streptococci of the c, c185, c160 and c85 series, give a visible signal for the concentration of 100 ng, the intensity of the signal being about 10 times weaker than that obtained for Streptococcus pneumoniae at the same concentration.
These results therefore demonstrate the lack of specificity respectively of the probes hexB-S.sub.7 and ami-S.sub.2 for Streptococcus pneumoniae, within the genus Streptococcus since a partial, but nevertheless significant, hybridization is detected with the species Streptococcus oralis, the closest species to Streptococcus pneumoniae and Streptococcus mitis. These probes are therefore unsatisfactory for the production of a selective test for detecting Streptococcus pneumoniae among other bacterial species which are most closely related.
In conformity with the publication by A. FENOLL, J. V. MARTINEZ-SUAREZ, R. MUNOZ, J. CASAL and J. L. GARCIA, Eur. J. Clin. Microbiol. Infect. Dis., 9 (June 1990) 396-401, other research studies led to the preparation of a hybridization probe (pCE3) for the genomic DNA of Streptococcus pneumoniae which is a 650-base pair fragment isolated from the lyt A gene, the latter encoding the N-terminal end of the streptococcal autolysin, amidase.
This probe was tested on 44 streptococcal strains among which 27 were identified as atypical streptococci strains and the other 17 as strains of Streptococcus viridans, based on conventional identification tests.
Although this probe provides a solution to the problem of the identification of Streptococcus pneumoniae, and in particular among atypical streptococci, it has, nevertheless, two disadvantages:
the probe used is a 650-base pair fragment and its production industrially is therefore not easy, PA1 and, in particular, the specificity of this probe is entirely linked to the presence of the lyt A gene, of which the copy in the genomic DNA of Streptococcus pneumoniae is unique; therefore, it will not be able to detect or identify a strain of S. pneumoniae from which the lyt A gene has been deleted; furthermore, this small number of copies is a disadvantage for the detection by direct hybridization and for the amplification of the target DNA.